ATP_ADAPT_LOW_ENERGY Report Summary

Final Report Summary - ATP_ADAPT_LOW_ENERGY (Adaptations of the ATP synthesis machinery in bacteria and archaea to conditions of extremeenergy limitation in the deep subsurface)

There are communities of microorganisms living in the deep marine subsurface under conditions of extreme energy limitation that have only recently become known to science. The original goal of the “ATP_adapt_low_energy” project was to determine how ATP was generated under such conditions of energy limitation, in particular adaptations of the ATP synthase protein complex. Since the project was first conceived, however, new information shifted the project’s focus: the first accurate measurement of volatile fatty acid concentrations (the electron donor that drives microbial metabolism) in the deep marine subsurface showed that available Gibbs free energy was higher than first assumed, and unlikely to place selective pressure on microbes’ ATP synthesis machinery. This gave the project two new foci: 1) use of the metagenomic dataset collected as part of the project to identify key differences in potential metabolism between microbes in energy-starved ice age sediments and energy-rich Holocene sediments, and to determine whether these differences are a result of climatic differences at the time of sediment deposition or as a response to changing in situ conditions and 2) use of a different metagenomic dataset produced during another parallel project at the Center for Geomicrobiology to investigate the rate-limiting step in organic matter degradation under extreme energy limitation in the light of newest discoveries, namely the breakdown of polymeric organic matter into monomers suitable for uptake and degradation by microorganisms.

For the project’s first new focus, we used metagenomes from sediment collected from down to 85 metres below sea floor during the Integrated Ocean Drilling Program Expedition 347, "Baltic Sea Paleoenvironment". These were used to analyse how the composition of buried microbial communities differed to conform to altered environmental conditions at depth. The sediments varied in age, organic carbon content, porewater salinity, and other parameters that reflect the changing Baltic environment from the last ice age and throughout the Holocene. We found microorganisms capable of energy conservation by fermentation, acetogenesis, methanogenesis, anaerobic oxidation of methane, and reductive dehalogenation. Glacial sediments showed a greater relative abundance of genes encoding enzymes in the Wood-Ljungdahl pathway and pyruvate:ferredoxin oxidoreductase than Holocene sediments. Relative abundance of genes conferring salinity tolerance was found to correlate with the present salinity, even in deep late-glacial sediment layers where salinity has increased since the sediment was deposited in a freshwater lake >9,000 years ago. This suggests that deeply buried and isolated sediment communities can slowly change in composition in response to geochemical changes that happen long after deposition. This discovery is important for making sense of long-term shifts in slow-growing microbial communities under extreme energy limitation, and shows that microbial communities existing under such harsh conditions can change in response to new environmental conditions and are not “locked-in” at the time the microbial community is founded. Researchers hoping to understand similar energy-limited environments, including those associated with marine oil and gas deposits and extraterrestrial deep subsurface environments, will find this knowledge useful.

The second new focus was on the degradation of biopolymers abundant on the seafloor, including carbohydrates and proteins. We now think that the extracellular degradation of these polymers is a rate-limiting step in the degradation of organic carbon in marine sediment, and therefore the step that defines these environments as energy limited and facilitating extremely slow growth by microorganisms. We used a metagenomic dataset from Aarhus Bay to identify the microorganisms responsible for extracellular polymer degradation, first by reconstructing genomes of individual bacteria based on sequence composition and variable coverage, and then by identifying genes encoding predicted extracellular polymer degradation enzymes (peptidases and carbohydrate-active enzymes) from these genomes to determine which bacteria play a key role in this rate-limiting organic matter degradation step. We found a large variation in the number of relevant enzymes between different taxonomic groups – bacteria belonging to certain phyla had no genes encoding extracellular polymer degradation enzymes, while genomes in other phyla, such as Planctomycetes, had over 100 of these genes. This shows that there is a distinctly uneven division of labor in between these different taxonomic groups and allows us to narrow down the important taxonomic groups that define the slow-growing nature of the deep subsurface environment.

This project also identified a newly named bacterial phylum as an important player in the degradation of detrital proteins. Calditrichaeota is a recently recognised bacterial phylum with three cultured representatives, isolated from hydrothermal vents. In the Aarhus Bay metagenomic dataset we identified two previously unknown examples of this phylum, allowing us to expand the phylogeny and ecology of this novel phylum with metagenome-derived and single-cell genomes from uncultivated bacteria previously not recognised as members of Calditrichaeota. Using 16S rRNA gene sequences from these genomes, we then identified 322 16S rRNA gene sequences from cultivation-independent studies that can now be classified as Calditrichaeota for the first time. This dataset was used to re-analyse a collection of 16S rRNA gene amplicon datasets from marine sediments showing that the Calditrichaeota are globally distributed in the seabed at high abundance, making up to 6.7% of the total bacterial community. This wide distribution and high abundance of Calditrichaeota in cold marine sediment has gone unrecognised until now. All Calditrichaeota genomes show indications of a chemoorganoheterotrophic metabolism with the potential to degrade detrital proteins through the use of extracellular peptidases. Most of the genomes contain genes encoding proteins that confer O2 tolerance, consistent with the relatively high abundance of Calditrichaeota in surficial bioturbated part of the seabed and, together with the genes encoding extracellular peptidases, suggestive of a general ecophysiological niche for this newly recognised phylum in marine sediment. This new view of a hitherto overlooked bacterial phylum is important for researchers hoping to understand organic carbon turnover in the shallow marine subsurface, and the resulting impact on global element cycling and its environmental consequences. Moreover, there are potential industrial uses for Calditrichaeota-derived extracellular protein degradation enzymes, as these appear to be active at a wide range of temperatures and oxygen concentrations.